The Minichromosome Maintenance Complex Component 2 (MjMCM2) of Meloidogyne javanica is a potential effector regulating the cell cycle in nematode-induced galls

Root-knot nematodes Meloidogyne spp. induce enlarged multinucleate feeding cells—galls—in host plant roots. Although core cell-cycle components in galls follow a conserved track, they can also be usurped and manipulated by nematodes. We identified a candidate effector in Meloidogyne javanica that is directly involved in cell-cycle manipulation—Minichromosome Maintenance Complex Component 2 (MCM2), part of MCM complex licensing factor involved in DNA replication. MjMCM2, which is induced by plant oxilipin 9-HOT, was expressed in nematode esophageal glands, upregulated during parasitic stages, and was localized to plant cell nucleus and plasma membrane. Infected tomato hairy roots overexpressing MjMCM2 showed significantly more galls and egg-mass-producing females than wild-type roots, and feeding cells showed more nuclei. Phylogenetic analysis suggested seven homologues of MjMCM2 with unknown association to parasitism. Sequence mining revealed two RxLR-like motifs followed by SEED domains in all Meloidogyne spp. MCM2 protein sequences. The unique second RxLR-like motif was absent in other Tylenchida species. Molecular homology modeling of MjMCM2 suggested that second RxLR2-like domain is positioned on a surface loop structure, supporting its function in polar interactions. Our findings reveal a first candidate cell-cycle gene effector in M. javanica—MjMCM2—that is likely secreted into plant host to mimic function of endogenous MCM2.


Materials and methods
Nematode growth, extraction and sterilization of eggs. Meloidogyne javanica was propagated for 4-6 weeks on tomato plants (Solanum lypopersicum cv. Avigail 870) grown in a glasshouse under a 16 h:8 h, light:dark photoperiod at 25 °C. Roots were washed and cut into segments, macerated in 0.05% (v/v) sodium hypochlorite (NaOCl) in a Waring Commercial Blender, 800G, at 22,000 rpm for 3 min, and subjected to centrifugal flotation as described by Hussey 20 to extract the nematodes eggs. The supernatant, containing the eggs, was poured onto a 30-µm sieve, and the eggs were washed with tap water and collected in 0.01 M MES buffer (Sigma-Aldrich, St. Louis, US). Nematode eggs were sterilized as described by Jansen van Vuuren and Woodward 32 , then collected and transferred onto a 30-µm sieve in a petri dish with 5 ml 0.01 M MES buffer. The petri dish was then placed in a growth chamber at 26 °C under dark conditions till hatching (5-6 days). All described experimental research on plants material was conducted under institutional and international guidelines and legislations.
Total RNA extraction from five M. javanica developmental stages. M. javanica eggs, freshly hatched preparasitic J2s (ppJ2s), parasitic J2s (pJ2s) 12 h after inoculation, three-to four-stage juveniles (J3-4s) and mature females were collected for total RNA extraction. The eggs and ppJ2s were collected right after sterilization. All other parasitic stages were isolated from the roots of in vitro-grown plants. Seeds of tomato cv. Avigail 870 were sterilized by soaking in 1.4% NaOCl for 10 min, washed three times with sterile water for 5 min, and then plated on standard-strength Gambourg's B5 medium salt mixture (Duchefa, Haarlem; The Netherlands), supplemented with 2% (w/v) sucrose and solidified with 0.8% (w/v) Gelrite agar (Duchefa) as described earlier by Iberkleid et al. 33 . Seeds were kept in a growth chamber at 26 °C for 3 d in the dark, and then transferred to a 16 h:8 h, light:dark photoperiod (120 µmol m −2 s −1 ). Two weeks after germination, tomato root segments were subcultured by placing one root piece per new Petri dish (90mm) (Miniplast, M.P. Hefer, Israel) containing Gambourg's B5 medium salt mixture for an additional week at 26 °C under dark conditions before nematode inoculation. Plates containing tomato roots were inoculated with 300 sterile ppJ2s; 12 h later, the typical thick hairy areas of root material (0.5-1 cm roots tissues), indicating nematode penetration, were collected into a 1.5-ml tube (~ 50 mg). For later time points, galls were collected 15 days after inoculation (DAI) for J2s and J3-4 stages, and 28 DAI for females without egg masses (harvesting ~ 50 mg root tissues for each stage and time point). RNA extracted from uninfected roots was used as a negative control. All samples were immersed in liquid nitrogen and stored at − 80 °C before RNA isolation.
Nicotiana benthamiana plant growth and Agrobacterium tumefaciens culture preparation. N.
benthamiana seedlings were grown in a glasshouse at 25 °C, under a photoperiod regime of 16 h:8 h, light:dark for 3-4 weeks, and A. tumefaciens strain GV3101 was used for all infiltrations. A. tumefaciens was transformed by using the freeze and thaw method 39 . The transformed A. tumefaciens was grown over 2 nights at 28 °C, with shaking at 180 rpm in LB medium including 10 mg ml −1 rifampicin, 100 ml −1 spectinomycin (for the construct pK7FWG2,0:MjMCM2) and 50 ml -1 kanamycin (for the organelle marker constructs), to an optical density at 600 nm (OD 600 ) of 0.6-1.0. From each culture, 1 ml was transferred to a new 50-ml tube containing 5 ml of LB medium with the same antibiotics, to an OD 600 of 0.4 (c. 4 h). The bacterial culture was then resuspended in 10 ml MMAi medium for 2 h at 28 °C with shaking at180 rpm, followed by centrifugation for 3 min at 1000 g, then resuspended in 1 ml of MMAi for 1 h at room temperature. The abaxial side of N. benthamiana leaves was infiltrated with transformed A. tumefaciens at a 1:1 ratio of pK7FWG2,0:MjMCM2 and different mCherry organelle markers. Two plants (about 9 leaves) were infiltrated for each experiment and the same experiment was repeated twice. Free-eGFP control (empty pK7FWG2,0) was used for the comparison (Supplementary Fig. S4). After infiltration, plants were transferred back to the glasshouse. Images were acquired 48 h after infiltration, using a Leica SP8 laser scanning microscope equipped with solid-state lasers with 488 and 552 nm light under a HC PL APO CS2 63X/1.2 water immersion objective (Leica) and Leica Application Suite X software (LASX). GFP and mCherry emission signals were detected with HyD (hybrid) detectors ranging from 500 to 530 nm and 580 to 650 nm, respectively.
In planta overexpression of MjMCM2. To overexpress MjMCM2, Rhizobium rhizogenes ATCC 15834 was transformed with pK7FWG2,0:MjMCM2 construct following the protocol described by Ron et al. 40 . Cotyledons of 10-d-old tomato cv. Avigail 870 seedlings were soaked for 2 h in a 50-ml tube containing 5 ml LB with www.nature.com/scientificreports/ 10 mg ml -1 rifampicin and 100 mg ml −1 spectinomycin and respective Rhizobium strain at optical density of 0.5 at 600 nm as described by Ron et al. 40 . These samples were then dried on autoclaved filter paper and plated on standard-strength Gambourg's B5 medium salt mixture, supplemented with 2% sucrose and solidified with 0.8% Gelrite containing 50 mg ml −1 kanamycin and 300 mg ml −1 timentin. The cotyledons were kept in a growth chamber at 26 °C in the dark. Hairy roots emerging from the cotyledons were transferred to Gamborg's B5 medium containing 0.8% Gelrite and 50 mg ml −1 kanamycin as described by Chinnapandi et al. 41 . The presence and expression of transgenic tomato hairy roots was confirmed by genomic PCR and this line was named MjMCM2 OE . For that purpose, total genomic DNA was isolated from the MjMCM2-overexpressing tomato roots and control line using cetyltrimethylammonium bromide (CTAB) method described by Goetz et al. 42 . 50 ng DNA was used to confirm MjMCM2 transgenic lines with forward 5′-ATG TAT GCT ATA CGA AGT TAT TAC G-3′ and reverse 5′ AGC AAT TGT TTT GAC AAT -3′ primers, which gave 2400-bp amplicon size. PCR reaction was performed as follows: heating to 94 °C for 3 min, 35 cycles of 94 °C for 30s, 60 °C for 30s and 72 °C for 2.5 min,followed by a final extension for 2 min.

Bioinformatics analysis. Classical nuclear-localization signals or known conserved motifs and domains
were predicted using Motif Scan (http:// myhits. isb-sib. ch/ cgi-bin/ motif_ scan/). Signal peptide sites were predicted using the SignalP 4.1 server (http:// www. cbs. dtu. dk/ servi ces/ Signa lP/; 43 . Subcellular location of effectors was predicted using WoLF PSORT (https:// www. gensc ript. com/ wolf-psort. html 44 49 : ^\w{10,40}\w{1,96}R\wLR\w{1,40}EER,2. to find a motif as part of the REGEX search that does not necessarily include a signal peptide and in addition, identifies effectors with the canonical W-Y-L motif found in RxLR proteins 50 , we implemented the custom script : regex.search (seq=ORF, motif = ",3. as part of the RGEX search, we used a pattern that searches for the RxLR motif in a greater area of the protein and also includes the modified regular expression pattern from Haas et al. 49

Results
In silico structural analysis of MjMCM2 and transcript abundance. In our previous transcriptomic study 25   MjMCM2 transcripts are localized to ppJ2 esophageal glands. FISH was used to study the spatial location of MjMCM2 in segmented freshly hatched M. javanica ppJ2s. We used a Cy5 probe on fixed ppJ2s following exposure to 9-HOT and as a control, those that were not exposed to the oxylipin. A strong signal was only observed in the esophageal gland of ppJ2s treated with 9-HOT and no signal was detected in ppJ2s that were untreated (Fig. 3b). MjMCM2 expression in the nematode secretory gland strongly suggests its potential secretion as a nematode effector during parasitism (Fig. 3a).
In planta subcellular localization indicates that MjMCM2 is targeted to the plasma membrane and the nucleus. To determine the effector's target site once delivered into the plant cell, we expressed the MjMCM2:eGFP construct under the control of the CaMV-35S promoter in N. benthamiana leaf epidermal cells together with endoplasmic reticulum (ER-Rb; 35S::mCherry-HDEL), Golgi apparatus (GmMan1-RFP) and cytoplasmic membrane (aquaporin PIP2A:RFP) markers (Fig. 4). MjMCM2 co-localized with the endoplasmic reticulum and Golgi network, as seen by the yellow fluorescence ( Fig. 4a-c,d-f, respectively). MjMCM2 signal was also observed in the plasma membrane, as well in the nucleus of some leaf epidermal cells (Fig. 4ag-i,j-l, respectively), as partly predicted by WoLF PSORT and LOCALIZER in silico (Fig. 4b). Free-eGFP control infiltrated leaves revealed a very faint and diffusive signal as observed in Fig. S4.   To further analyze the impact of MjMCM2 on regulation of feeding-site development, we conducted morphological and cytological analyses of sectioned galls 28 DAI. Gall sections were stained with DAPI to visualize morphological changes in the feeding sites and distribution of nuclei (Fig. 6). Galls overexpressing MjMCM2 showed apparently larger GCs (Fig. 6a,a') that were more densely filled with larger nuclei harboring prominent chromocenters compared to the control (Fig. 6b,b'). More visible nuclei per section upon MCM2 overexpression (Fig. 6c) suggests higher mitotic activity in these GCs.
In general, the RxLR motif is defined by the sequence Arg-x-Leu-Arg, where x is any amino acid, and in some cases it is followed by an acid-rich DEER motif (Asp-Glu-Glu-Arg). Sequence analysis of MjMCM2 indicated that it does not contain a signal peptide for secretion, or any transmembrane domain. However, further investigation revealed the presence of an acidic region characterized by a SEED (Ser-Glu-Glu-Asp) motif followed by an  1.1.scaf04253), and these were aligned using ClustalW program 55 . Sequence alignments indicate a conserved SEED-RxLR-like motif in nematodes belonging to the Meloidogyne family (Fig. 7).
MjMCM2 3D homology modeling. Homology modeling is a standard structure-prediction method that contributes to understanding the relationship between protein structure and function. To investigate the molecular bases of the different functions elicited by the second RxLR, the 3D structure of MjMCM2 was predicted by homology modeling, using the SWISS-MODEL server 51 . The Local Quality plot (https:// swiss model. expasy. org/ docs/ help# qmean) showed that the predicted structure contains mainly two large regions of good local alignment (QMEANDisCo > 0.60), where the second region contains the second RxLR (Supporting Information Fig. S2, inset shows sequence of residues surrounding the second RxLR). Secondary-structure representation demonstrated that the second RxLR is positioned on an exposed loop (Supporting Information Fig. S3a). Surface representation of the molecular model showed that the RLR residues of the second RxLR motif were on the surface in a polar environment (Supporting Information Fig. S3b, blue indicates polar), inside the region with the high local alignment score (QMEANDisCo) relative to the template used for the homology modeling (Supporting Information Fig. S3a).

M. javanica genome mining for sequences encoding proteins carrying a RxLR-like motif. To
further study the occurrence of RxLR-like motifs in the M. javanica genome, we conducted a comprehensive de novo gene prediction of RxLR-like motifs on M. javanica ORFs using the effectR program developed by Tabima and Grünwald 48 as a genome mining tool. This program enables rapid and reproducible prediction of effectors in oomycete genomes, or with custom scripts in any genome. The effectR package relies on a combination of regular expression statements and hidden Markov model approaches to predict candidate RxLR motifs 48 .
For the genome search, three scripts were used (Supporting Information Table S1): a strict motif pattern-the conserved RxLR-DEER motif (*RxLR), resulting in the identification of 217 candidate proteins, of which only two proteins carried a canonical signal peptide; the motif relax mode-**RxLR, resulting in 1305 candidate www.nature.com/scientificreports/ proteins, of which only 12 proteins carried a canonical signal peptide; and the motif relax2 mode-***RxLR, resulting in 2525 candidate proteins, of which 54 carried a canonical signal peptide. A conserved and complete RxLR-DEER-like motif was found on two different scaffolds encoding superoxide dismutase (SOD), and one encoding α-amylase (Supporting Information Table S2). Degenerate RxLR motif was also found in cell walldegrading enzymes, such as the arabinanase gene required for polysaccharide degradation 56 , and the glycoside hydrolase (GH) that catalyzes the hydrolysis of the glycosidic linkage of glycosides 57 (Supporting Information  Table S2).

Discussion
The establishment of galls induced by RKNs involves a number of alterations in the plant host root that are essentially driven by secreted molecules. These molecules likely cause the dedifferentiation of vascular cells into GCs, forming a feeding site upon which the nematode depends to lay eggs that will hatch into new infective juveniles. These GCs are highly dependent on cell-cycle activity leading to multinucleation via acytokinetic mitosis, followed by nucleus enlargement caused by the action of the endocycle. Stimulation of the host's cell-cycle machinery may be potentially controlled by candidate secreted effectors, as part of the nematode's manipulation of host gene expression, but these and their targets remain to be identified 11,58 . For DNA synthesis during the S phase, genes involved in mitosis as well as the endocycle, such as ORC1-6, MCM5, CDT1a,b and CDC6, have been found to be expressed in GCs, characterized by the prominent DNA synthesis needed for their cell-cycle machinery 8 . Several potentially secreted nematode proteins-a CDC48-like protein, a ubiquitin, and a SKP1-like protein-with possible roles in cell-cycle regulation have been identified in silico 59 . However, so far, their role in cell-cycle regulation during feeding-site formation and development has not been demonstrated. Herein, we uncover the first candidate effector gene, MCM2 of M. javanica, which is likely secreted and may directly or indirectly affect the host cell cycle. MjMCM2 might act by mimicking the function of www.nature.com/scientificreports/ plant-endogenous MCM2 as a licensing factor, facilitating the transformation process of root cells into large multinucleate feeding cells.
The M. javanica genome has multiple copies of MCM2 genes. M. javanica reproduce by mitotic parthenogenesis, as they have a polyploid genome with highly divergent genome copies, apparently resulting from hybridization events, ploidy changes and chromosomal fragmentation 60 . Homologous gene copies generally exhibit different gene-expression patterns 61 . M. javanica has 26,917 predicted genes and 944 pseudogenes (BioProject PRJNA340324), despite the latter's exhibit general homology to known genes, they are not functional. In addition, transposable elements occupy ~ 50% of the genome, providing genome plasticity 60 . These previous observations might explain the various MCM2 gene copies found in M. javanica. MjMCM2 (M.Javan-ica_Scaff2271g021798) lacked a canonical signal peptide and transmembrane helices. As previously reported, many secreted effectors have been found to lack a predicted signal peptide 62,63 . In addition, in silico approaches have limitations: accurate N-terminal annotation is critical for signal peptide identification, signal peptides are highly heterogeneous, and some of them are indeed difficult to predict 64,65 . MjMCM2 is expressed in the nematode esophageal gland and is upregulated during parasitic stages. To determine whether MjMCM2 expression plays a role during nematode parasitism, we assessed its organellar location and its expression during the nematode's parasitic stages. FISH clearly localized MjMCM2 transcripts to the ppJ2 esophageal gland, the main secretion organ of PPNs. This strongly supports the notion that MjMCM2 is a secreted protein. Furthermore, qRT-PCR strongly suggested that MjMCM2 expression is not only predominantly induced upon nematode penetration into the plant root, but is also continuously expressed during feeding-site establishment, supporting that MCM2 might be positively affecting the repeated mitosis cycles occurred in the nematode feeding sites.
MjMCM2 co-localizes with the plasma membrane, endoplasmic reticulum and nucleus of N. benthamiana leaves. Localization of MjMCM2 in N. bethamiana leaves revealed its potential function in the plant host during nematode infection. Thus, an assay was performed to determine the cellular compartments that this protein might target when secreted in GCs during parasitism. Subcellular localization of our potentially secreted effector MjMCM2 fused to eGFP in N. benthamiana leaves was tracked along the plasma membrane and in the nucleus as predicted in silico. MjMCM2 localization in the endoplasmic reticulum and Golgi apparatus suggested that this protein might enter the cell's secretory pathway, and indicated its recognition by the plant machinery as described previously by Jaouannet et al. 66 , when studying the Mi-CRT effector localization.
In addition, we must consider that some effectors must undergo modifications to be targeted to their subcellular localization, as well as for their biological activity 67,68 . Thus post-translational modifications might occur after MjMCM2 secretion so that it can properly perform its function, as a complex that is imported into the nucleus to be assembled into a pre-replication complex at the M/G1 cell-cycle stage 69,70 . Moreover, nematode effectors that are targeted to the plant nucleus are predicted to be involved in the regulation of the plant cell cycle. Since GCs are also connected to neighboring cells by plasmodesmata 71 , it could be speculated that secreted proteins such as MCM2 might diffuse to, and be involved in cell-cycle activation in other gall cells in the vascular tissue where the gall is located. Moreover, the change observed in nuclei appearance in the overexpressing galls might be the result of increased mitotic cycles. In addition, enlarged nuclei harboring multiple large chromocenters suggests increased endoreduplication, likely facilitated by MjMCM2 overexpression. Changed nucleus morphology in GCs is consistently seen during functional studies of cell-cycle genes [12][13][14][15]72 . Furthermore, Kondorosi and Kondorosi 73 , have shown that in Arabidopsis the endocycle induce key S-phase genes such as ORC, CDC6, CDT1 and MCM genes. Interestingly most of these genes are somewhat expressed in galls 74 , supporting their implication in typical endocycle route during nematode feeding site establishment and maintenance. Findings observed here, are also in a good agreement with previous studies demonstrated the early upregulation of key components of the core cell cycle machinery in nematode feeding sites (CDK,1, CDKB1,1, CYCA2,1 and CYCB1;1 as shown by promoter activity and transcript localization 3,11 . Given that MCM2-7 is targeted by several different kinases including CK2, cyclin-dependent kinases (CDK) 75 , their co-occurrence in nematode feeding sites might ensure its modulation.
The process of identifying genes encoding candidate effectors in the nematode genome has become an important tool, as these proteins are directly involved in pathogenicity.
We know that some effectors are secreted through non-classical secretion pathways. We also know that RKNs secrete some effectors from their stylets into the apoplast 76 , and yet data suggest that these effectors are translocated to, and function inside the plant cells. There is a need to study RKNs for non-canonical secretion and effector translocation into host cells. This led us to look at other pathogens and their effectors. For example, there are oomycete effectors containing a signature RxLR motif 77 . This motif is implicated in translocation of the pathogen proteins into host cells 78,79 . Interestingly, signal peptide-lacking RxLR effector proteins have been shown to be secreted unconventionally 63,80 . In the past, there has been limited genetic information for PPNs, and initial scans of PPN genomes concluded that there were no effectors with RxLR or similar translocation motifs 81,82 . However, more recent work has identified RxLR motif-containing genes in the cyst nematode Heterodera avenae. In a transcriptome analysis comparing preparasitic to parasitic H. avenae, 61 transcripts were identified that encoded proteins predicted to have secretory peptides and an RxLR motif. These proteins were identified as putative cyst nematode RxLR effectors 83 . Herein we provide the first evidence of an effector with conserved RxLR motifs being present and functioning in PPNs.
Our observation of two RxLR-like motifs along the amino acid sequence of MjMCM2 raises substantial questions about its function in translocation to the host plant. One such question is whether the two motifs found in www.nature.com/scientificreports/ MjMCM2 potentially exert similar effector translocation behavior as the single RxLR-motif in oomycetes 49,84,85 . The RxLR motif was originally identified by comparing sequences of effectors from Hyaloperonospora arabidopsidis, Phytophthora infestans and Phytophthora sojae 86 . Following intensive studies, the RxLR-motif is now considered important for translocation of oomycete effectors into plant cells 79,87 . Localization of MjMCM2 to the nematode secretory glands and its overexpression in planta strongly suggest its secretion into the host root. While no RxLR effector proteins have been found in RKNs 88 , herein we suggest the occurrence of a degenerate RxLR-like motif displayed as a SEED--RxLR motif twice in the MjMCM2 protein. Our in silico analysis encompassing several nematode species demonstrated the first RxLR-like motif in all available MCM2 amino acid sequences. However, the second RxLR-like motif observed here was solely found in Meloidogyne spp., it was absent in other Tylenchida PPNs and in other free-living Rhabditida nematodes. Molecular homology modeling predicted the position of the second RxLR-like domain on a surface-residing polar loop. As surface loops have been suggested to participate in protein-protein interactions 89 , this result supports its postulated interactive function with other proteins. Similarly, two adjacent RxLR motifs were found in the effector protein Avr1b from Phytophthora sojae, although the RxLR-motif was found not to be essential for avirulence function 79 . Future studies should focus on the functional aspects of these RxLR-like motifs, particularly through direct mutagenesis. Further mining of the M. javanica genome should be performed using the effectR package, designed to predict effector proteins, including other RxLR effector proteins. Use of this approach by Tabima and Grünwald 48 resulted in the discovery of several genes in the M. javanica genome, e.g., SOD and α-amylase, whose secretion and function have been studied during parasitism by other PPNs 90,91 . Similarly, other cell wall-associated proteins, such as GH which catalyzes the hydrolysis of the glycosidic linkage of glycosides 57 , the 5-L-arabinanase 1 gene that is required for polysaccharide degradation 56 and a laminin-like protein that interacts with receptors anchored in the plasma membrane of cells adjacent to basement membranes 92 , have all been shown to carry degenerate RxLR-like motifs.

Concluding remarks
Overall, our study places MjMCM2 as the first candidate effector that might directly affect the mitotic and endoreduplication cycles in RKN-induced GCs, during gall genesis. Our localization studies strongly suggest that this protein is secreted by the nematode and that it can potentially be transported to the nucleus after possible structural modifications in the endoplasmic reticulum and Golgi bodies. Overexpression data suggest the importance of this MjMCM2 for parasitism, suggesting the use of these data for application in crop species, for e.g., by expressing double-stranded MjMCM2 in planta, then silencing it in the nematode. The discovery of two RxLR-motifs in this MjMCM2 protein has to be further investigated to determine whether MjMCM2 translocation into the plant follows a non-conventional pathway. Furthermore, it would be interesting determine the relevance and function of this repeated motif. These findings might contribute to distinguishing RKNs as that have the ability to induce multinucleate feeding cells and help us understand the molecular mechanisms governing plant-nematode interactions.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.